鸡传染性法氏囊病病毒VP2基因的克隆及序列分析

用SPF鸡胚成纤维细胞繁殖鸡传染性法氏囊病病毒(IBDV)J1C7毒株。用纯化的病毒颗粒提取RNA基因组。根据已报道的IBDV JD1株的VP2序列设计引物,利用反转录—聚合酶链式反应(RT-PCR)进行扩增,将获得的1 359 bp片段克隆于pMD 18-T载体上。经过限制性酶切及PCR鉴定后,得到阳性重组质粒。对VP2基因进行全序列测定表明,克隆的VP2基因长1 359 bp,编码452个氨基酸。J1C7株VP2蛋白高变区内两个亲水区与其他标准Ⅰ型毒株完全相同,而且具有弱毒疫苗株的特征性氨基酸:222P、256V、279N、284T、294L、299N,七肽区的氨基酸序列为SWS ARGS,因此,从分子水平上推断本研究的J1C7株为标准Ⅰ型毒株,且属于弱毒疫苗株。与已报道的IBDV VP2基因的氨基酸序列进行比对,结果表明,本研究的J1C7株与来自欧洲和中国的弱毒疫苗株有密切的亲缘关系。

Cloning and sequence analysis of VP2 gene of infectious bursal disease virus

The Infectious bursal disease virus J1C7 strain was propagated in 10-day-old specific-pathogen-free chicken embryo fibroblast.Viral dsRNA was extracted and purified.A pair of primers was designed according to the VP2 gene sequence of IBDV JD1 strain reported previously.The VP2 fragment in length 13 59 bp was amplified by RT-PCR,the products were cloned into pMD18-T vector.The recombinant plasmid was identified by restriction enzyme cut and PCR.The sequencing of VP2 gene of J1C7 indicated the VP2 gene was 1 359 bp.The amino acids of two hydrophilic regions in the variable region of VP2 were wholly conserved with standard Ⅰtype strain.In addition,IBDV J1C7 strain has the typical amino acids which are belonged to attenuated IBDV strains,it was suggested that IBDV J1C7 strain is standard Ⅰtype strain which belong to the attenuated IBDV.Compared with several other strains,the amino acid alignment report and phylogenetic tree revealed that J1C7 was most closely related to the attenuated IBDV strains which come from Europe and China.